CA2098105C - Oligoside derived from an antigen polyoside obtained from a pathogen - Google Patents

Abstract

An oligoside derived from an antigen polyoside obtained from a pathogenic agent, a method for its preparation, and its use particularly as a vaccinal agent. The oligoside is prepared by oxidation-reduction depolymerisation reaction.

Description

~,: ~: '~~ xy ~ 4 ~; .

"Olir ~ osida derived from an antiKénicrue uolvosida of a sweating uathoxene "
The present invention relates to an oli ~ oaide derived from a polyoaide antigenic from a pathogen; its preparation process and its use in particular as vaccine agent.
Bacteria as well as fungi such as yeast incorporates polyaids into their surface structure. So the wave majority of bacteria are covered with a polysaccharide exudate which is attached to the bacteria in a stronger or weaker way but which is not at properly speaking an envelope. This exudate is called a capsule or glycocalyx. Besides the external member of grem-negative bacteria East among others consisting of lipopolysaccharide (LPS). Finally, polyeaccharidas are also found in the wall of mushrooms. Polyoeidea cases are in makes surface antigens that induce an immunological response in an infected mammal.
Such polyoeidea are formed on the basis of repeating units, in which components and bonds are defined and which are each characteristics of the fungal or bacterial species considered. These units r ~ p ~ t1t1V88 COIItlenllent 188 épltOpea, the decisive structures are da antigenicity. As an illustration, we have reported in Figure I, diïférents types of repeating units from polyoaidea capsulais as or from lipopolysaccharide. Repeating units often contain functions very labile such as for example phosphate functions, present in the backbone of the molecule or in a branched position as well as, for example 0-acetyl and pyruvatea groups.
A polysaccharide is actually made up of a set of polymer molecules containing osidic units in number which can vary from one molecule to a few other. Therefore, a polysaccharide cannot be described in terms of weight molecular than by an average molecular weight. In the case of a homopolyoside, the sugar units are monosaccharides. In the case of a heteropolyoeide the osidic units form a chain of constituents linked together in a definite way.
SHEET DIE RE1V ~ PLA ~ CEt ~ AENT

2 Regarding the average molecular weight of a polysaccharide, it can be assessed by gel filtration. The molecular weight is then expressed in terms of constant elution (KD) or dextran equivalent (EqD) according to the method described by Granath et al, J. Chrom. (1967) ~. 69 that one summarized as follows A polysaccharide in solution is analyzed on a column exclusion-diffusion chromatography gel which separates elements according to their molecular weight. This type of gel is commercially available, for example under the names of Sephadex * (Pharmacia), Bio-Gel * (Bio-Rad), Fractogel * (Toyo-Soda), Sepharose * (Pharmacia), and Sephacryl *
(Pharmacia). The gel fractionation area should be well obviously suitable for the range of molecular weights represented within the molecular population to be analyzed. AT
As an example, it is indicated that the gel columns of Sepharose 2BCL *, 4BCL * and 6BCL * filtration have a zone respective fractionation determined in dextran equivalent from 20,000,000 to 100,000; 5,000,000 to 20,000 and 1,000,000 to 10,000. Generally, an eluent is used saline, for example 0.2 M sodium chloride. One elution constant is calculated according to the following formula:
Ve Vo Vt - Vo in which:
Ve is the elution volume of the preparation considered; Vt is the total volume of the column; and Vo is the dead volume of the column.
By way of example, Figures 2a and 2b show the respective elution profiles of the Salmonella typbi polysaccharides and Streptococcus pneumoniae type 1 on a colomu ~
Sepharose 4BCL *. The polysaccharide of S. typbi is therefore composed of polymers with the highest molecular weights are * (trademarks) 2a not appreciable on Sepharose 4BCL *. Similarly, the polysaccharide of S. pneumoniae type 1 is mainly composed of polymers whose molecular weights vary from zero KD to 0.45. Through definition, the mean elution constant of the polysaccharide is appreciated at the point of intersection of the tangents of the slopes of the elution profile; either approximately, at the top of the elution profile. Thus, the mean elution constant of polysaccharides shown in Figures 2a and 2b is respectively zero and 0.04.
* (trademarks) .. wa ~;., ~ j; ,;
... 1;

3 On the basis of a standard range established using daxtr an, we can COllVertil uYle CO178 elution molecular weight expressed in EqD. AT
titi e For example, Figure 3 shows a standard range model.
Because of their antigenic power, polyoids, antigens of surface pathogens eg bacterial or fungal are good candidates as a vaccine agent. Vaccines with an extract polyoeidic have indeed proven effective in adults. However Phone is not the case in young children. In order to overcome this inconvenience major, it has been successfully proposed to link the polysaccharides covalently to pr Ote111e8, which gives the molecules thus obtained a character T-dependent and increased their immunogenicity.
For example, vaccines with basa da polysaccharides include vaccines to date against infections with Iv'eissarie menin ~ itidis groupa A, C, Y or W 135 to Streptococcus pmeumoniea, Hsemopfülus influenzea type B and Salmonella typhi.
Although the antigenic polyaids of pathogens are potentially interesting as immunogens, their use is revealed delicate due to the importance of their molar weight: average .ular which can reach several million dextran equivalent. In particular, when one wishes to use a polysaccharide in the form of a conjugate, that is to say coupled to a protein, we ran into technical problems that were difficult to solve.
During the coupling process, a gel or flocculate can form, thus making the conjugate difficult to sterilize by filtration.
In order to overcome this pitfall, it has already been proposed to reduce the size of poloids to make them easier to handle. Ultimately, we have recommended various cleavage methods. So there are for example well-established methods such as fragmentation by ultrasound or by hydrolysis in an acidic, basic or enzymatic medium. However, these methods of fragmentation are far from satisfactory. Indeed, they drive too often to the total or partial destruction of the characteristic epitopaa by REPLACEMENT SHEET

4 loss of essential labile chemical functions for antigenic specificity, such as phosphate groups acetyl and pyrurate.
Thus, acid or basic hydrolysis of the polysaccharide capsular of S. typbi or N. meningitidis group A eliminates the acetyl groups which are necessary for the specificity polysaccharide antigen. In addition the correct reacetylation of fragments obtained by hydrolysis is very difficult otherwise impossible.
On the other hand, with the method of fragmentation by hydrolysis, it is not always possible to obtain fragments of relatively homogeneous size, while the pharmaceutical practice requires homogeneous products, constant and standardized quality.
When it comes to ultrasonic fragmentation, we have for example shown that the phosphate groups polysaccharide terminals of H. influenzae type B are lost by ultrasonic treatment. This may raise concerns that phosphate groups in branched position on the chains polysaccharides are similarly eliminated during such a treatment.
In addition, although fragments of relative size-homogeneous can be obtained with ultrasound, obtaining fragments of sufficiently small size by the aux method ultrasound requires a very long processing time, which can cause among other disadvantages, deterioration rapid switchgear. Its effectiveness being limited, the application of ultrasound on an industrial scale is to exclude.
Finally, the use of depolymerization polysaccharide enzyme is limited to polysaccharides for which suitable enzymes are known. This disadvantage major however is inherent in the highly specific character enzymes vis-à-vis their substrate.
For the vaccine field, technical progress and important economic would be to be able to have a 4a depolymerization process which no longer presents the various disadvantages of the prior art methods. In in other words, it would notably be necessary to have a J
process of depolymerization of anti ~ enic polysaccharides which has a set of pr oprierties avanta ~ eusee by what - It makes it possible to obtain oli ~ osides having a number of units osidics reduced while oonser is worth the structural determinants essential of at least one epitope of the polyoeide to fr ~ lie.
- It is possible to use it for any structure polyoeidic anti ~ enique envisaged.
- It allows to obtain fragments of sufficient size homogeneous, and - It is low cost and very simple to . Implementation.
Surprisingly, we have now found that these objectives are reached by an oxidation-reduction depolymerization reaction.
So far, such a method, often referred to in language an ~ lsise under the acronym ORD, was implemented to fragment polysaccharides such as heparin, hyaluronic acid, dextran and DNA and it â fine totally different from those pouf followed by this request. It was ~ issait for example to reduce the viscosity of the products or, in the case of a linear molecule such as heparin to obtain small fragments which were known to have activity different anticoagulant. Of course, like the products to which this method applied were not of immuno ~ enic interest, the study of the integrity of the repeating units of the fragments obtained was not addressed.
On the other hand, there are many previous publications such that, for example, K. Uchida, Carbohydrate Research, (1988) 1-3: 89-99; H.
Uchiyama, Journal of Biolo ~ ical Chemistry, (1990) 265:? 753-7? 59; SW
Green The Carbohydrates -Chemistry and Biochamistry IB; Academic Prese FE1JlLLE ~ E REPLACEMENT

(1980): 1132; A. Herp, The Carbohydrates Chemistry and Biochemistry IB; Academic Press (1980): 1276; Kochetkov et al, Radiation Chemistry of Carbohydrates, Pergammon Press, Oxford, (1979) who describe the ORD reaction as destructive either phosphate groups, i.e. carbohydrate cycles by cleavage of carbon-carbon bonds in the cycle.
The overall impression that emerged from the state prior to the technique, the ORD reaction is generally recognized as destructive.
There was therefore nothing to suggest that the DSB would be better than for example the hydrolysis method or by treatment with ultrasound for the purposes sought, ie, conservation of epitopes.
This is why, before the present invention there had never been shown to be possible oligosides, whose structure of the repeating unit character ristic is preserved, by redox fragmentation of respective polysaccharides, in order to be able to use them as of active agent in formulations making it possible to reinforce 2o an individual's immune defenses.
We have now discovered that it is possible to implement the ORD method to obtain oligosides having kept at least one antigenic determinant of the polysaccharide of departure.
Furthermore, it was also found that the method of redox depolymerization applied to a polysaccharide antigenic allowed to obtain under good conditions completely satisfactory fragments from the point of view of uniformity of size, despite the fact that it is known that 30 the ORD reaction is a radical reaction cleaving the polysaccharide randomly.
Accordingly, the present invention provides a process for the preparation of an oligoside by depolymerization of a antigenic polysaccharide from a pathogen, including repeating units contain at least one chemical function labile, retaining the structure of repeating units with their labile functions, in which.
(i) subjecting said polysaccharide to a reaction of oxidation-reduction depolymerization, and (ii) the oligoside thus obtained is collected.
The present invention also relates to oligosides obtained by this method, having kept at least one antigenic determinant of the antigenic polysaccharide derived from the pathogen.
Those skilled in the art will understand that the labile function in question is preferentially chosen from groupings phosphate, acetyl and pyruvate.
By "oligoside" is meant a set of molecules:
each molecule having a reduced number of osidic units, by compared to the starting polysaccharide, and containing for example 4 500 units of sugar.
The average molecular weight of an oligoside is defined according to the rules explained above for a polysaccharide.
Generally, an oligoside according to the invention can have a average elution constant on a Sepharose 4BCL * column 0.2 to 1, in particular from 0.3 to 0.9, in particular from 0.9 to 0.8, for example from 0.6 to 0.7. In other words, such an oligoside has an average molecular weight of 200,000 to 2,000 EqD, in particular from 150,000 to 10,000 EqD, in particular from 60,000 to 30,000 EqD.
According to a preferred aspect of the present invention, the antigenic polysaccharide is a surface antigen from a pathogen which can be a fungus as well as a Gram-negative or Gram-positive bacteria, especially of the genus Staphylococcus, Streptococcus, Kle, bsiella, Salmonella, Escherichia, Neisseria, Shigella or Haemophilus.
The bacterial pathogen is for example Streptococcus pneumoniae, Neisser ~ a meningitzdis, Salmonella typbi or Xaerriophilus influenzae.
* (trademark) 7a The fungal pathogen can be yeast, plus particularly selected from the Candida genera, Cryptococcus, Hansenula, Lipomyces, Rhinocladiella and Rhodotorula.
For the purposes of the present invention, a reaction oxidation-reduction is carried out in the presence of at least one redox system which can be selected in particular among thiols eg, glutathione, cysteine; oxygen;
hydroquinone; multi-valence metal ions, eg, iron and copper; ascorbic acid, hydrogen peroxide; these the latter two being preferred.

: V ~ i ~~ '~' ~~ ii..w s The different experimental parameters that can influence the kinetics of the reaction ~ CG11C811trat1011 of the products, pH, temperature, time da reaction) can be easily determined by a person skilled in the art 6r ~
fallCtlall of the ietiquea characteristics of the starting polyacid, of the oxidio-J redüCtIUIl BeleCtIUIllle, and d6 the average size of the fra ~ mellts it wish get. When case fra ~ mellt8 Ballt deSt111é8 notamme2lt ~ de9 flIlS
VâCC1I181eS, the skilled person will take particular care to adjust the different parameters so as to obtain fragments of medium size suitable, that is to say retaining a ban power anti ~ enic and possibly adapted to the implementation of canju ~ ues; for example, from uli order of ~ r ardor as previously indicated. In a way ~ ener ale, the CGIldltla118 ré8Ctlan I'ie11e8 aptlmale8 can be determined11 IOrB d8 test6 of r tool. As an indication, however, specified in the examples below after experimental values allowing to obtain good results.
A polyoeide intended to be subjected to a process according to 111Velltlall can be extracted and purified by known methods, in particular by precipitation with alcohol or cetyltrimethylammonium bromide or by filter ation on Rel. For a review of these methods, we refer in par ticular to RL -Whistler, in Me.thodc in Carbahydrate Chemistry, (1965) I: 3-62.
The polyacid is advantageously prepared in aqueous solution at a concentration of 5 m ~ / ml. It is indicated that an advantageous concentration can vary from 0.5 to 50 m ~ / ml, in particular from 0.5 to 10 m ~ / mI. Such a solution 2 5 aqueous can be used as a starting product.
An oxidative-reducing system as defined above is added to the polysaccharide preparation all at once or continuously or discontinuous in a final weighted oxidation-reduction system polyoeide which can vary in particular from 0.01 to 10, in particular from 0.1 to 5, for example from 0.1 to 1.
A process according to the invention can be carried out at a pH of 3 to 8.5, in particular from 3 to 6.5. Likewise, the temperature of the reaction medium is not critical; it can in particular vary from 4 to 60 ° C., in particular of 18 to 40'C.
SUBSTITUTE SHEET

Ai ~ .i ~~~: ~ .. 'i ~.

The time necessary to achieve a desired fr a ~ mentation of the polyoeid is generally from 30 min to 1 h except in special cases; by exempt, for the polysaccharide Vi of S. typ ~~ i, the reaction time is significantly longer long. The reaction time can be adjusted according to the size of the fragment that we wish to obtain.
According to a particular embodiment, to an aqueous preparation of a polysaccharide ayatzt ulle CUIlCBIltratlOtl from 0.5 to 50 mg / ml, in particular 0.5 to 10 mg / ml, ascorbic acid is added to a final concentration of .
0.01 to 25 mg / ml, in particular 0.1 to 10 mg / ml. Naturally oxygen dissolved in the reaction medium is generally sufficient; if tel n'ctait not the case, one could inject me in the medium. In order to speed up the reaction, we prefer preferers, in the presence of a soluble salt of a metal with several degrees of oxidation, such as iron or copper. The or, the sella) metal) can (wind) be added (a) at a concentration of 1 ~ M to 100 mM, in particular from 1 to 10 mM.
According to an alternative embodiment to that set out above, we replaces ascorbic acid with hydrogen peroxide which can be added up to a concentration of 0.1 to 100 mM, in particular 0.5 to 10 mM, optionally in the presence of a metal salt.
An oligoside prepared by subjecting a polysaccharide to a reaction ORD depolymerization consists of a set of molecules, the mean elution constant is lower than that of the polysaccharide from which it is derived by fragmentation (the comparison is obviously implemented in using the same chromatography column). Oligoside can be isolated according to a conventional technique, for example by precipitation using of a suitable precipitating agent eg, acetone or alcohol, by filtration on membrane with an appropriate epar ation threshold, by chromatography exclusion-diifueion or ion exchange. Thereafter, we can also choose to use only because fractions of oligosida containing molecules having a constant elution equal to, or around the corset 3 5 of medium elution.

WO 93/07178 ~ PCT / FR92 / 00955 ~ ae, ~. ~ ~;, ~ ~~, ~ Wi; . . _ ~
Optionally, the oligoside according to the invention can be coupled by lisisoN covalent to a compound of peptide, protein or other nature organic polymer as by exempted the polyacrylate to form a conjugate capable of promoting the immunogenicity of the oligoside In particular in

5 a mammal. The conjugation partner can be a particular product111 bacterial, for example a toxin, toxoid because r espondante or a sub-unit of a multimeric toxin as well as a membrane protein, an aus-unit of a multimeric membrane protein or a cytoplasmic protein.
By way of example, U11, cites the toxin Pertussis, the toRIlle CllUlérlque, 18 tOAlIIC
10 tetanus and diphtheria toxin. These proteins can be extracted â
from the original bacteria or else be obtained recombinantly.
A coupling reaction, by covalent bond, of an oligoside with uli pal ~ te11a11 ~ e from CGlljuga18G11 so ~ li to get usable uli COnjugüé
e11 both that vaccine can be implemented in a conventional manner. We can by example show on the oligoside a function capable of reacting with a functional grouping of the conjugation partner. We can also do e react a bi-functional coupling agent with the oligoside and then with a conjugation partner, or vice versa. For a review of these various coupling methods, we refer in particular to WE Dick and M. Beurr and, in Conjuguates Vaccines, JM Cruse, RE Lewis Jr Eds, Contrib. Microbiol.
Immunol. Basel, Karger, (1989) 10: 48. Furthermore, the method of fr agmentation by. ôxydo-r éduction introduces reducing groups, especially in oligosides derived from the polysaccharides of S. pneumoniae type 6B and 19F, B. influanzaa and N. menin ~ itidis group A. This therefore allows to use the conjugation technique by reductive amination.
The immunogenicity of an oligoeide according to the invention can also be favored by using liposomes carrying a to which the oligosides are related covalently. Also promote immunogenicity, vectors, who retain the oligoside by incorporation using ionic forces or hydrophobic, such as for example cyclodextr ires, liposomes and iscoms.
The invention therefore relates to an oligoside as defined previously presented REPLACEMENT SHEET

'a wt, ~~. ~
~~~ y in various forms, especially in the form of a conjugate, or coupled to a carrier liposome, or incorporated into a vector. According to a preferred aspect of the present invention, the oligoside is in the form of a conjugate.
An oligoeide according to the invention is IlOtammellt useful for reinforcing the immune defenses of an individual, in mammals and in particular in humans, for example to prevent or mitigate the Effects of an infection induced by a pathogen eg, a bacterial infection or a yeast infection.
Mixtures of oligosides according to the derivative invention are also useful.
of different antigenic polyoids, as well as mixtures of an oligoside according to the 111VeI1tlU11 aV6C active agents other than an oligoside according to the 111ve21t10I1. Of such mixtures are suitable for the preparation of polyvalent vaccines.
Consequently, the invention also proposes - A macharic pharmaceutical composition which comprises, as active agent su at least one oligoside according to the invention; the composition is notably a vaccine;
- The therapeutic use of an oligoside according to the invention;
- A method to strengthen an individual's immune defenses eg, vaccination, which includes the act of administering, in quantity sufficient from a therapeutic point of view, to the monk an oligoside according to the invention, to a subject in need of a. such treatment eg, such VaCClIl8tlo11;
- The use of an oligoside according to the invention as an active ingredient in the preparation of a pharmaceutical composition intended to reinforce ~ an individual's immune defenses;
- A process for the preparation of a pharmaceutical composition such as previously defined, characterized by the fact that (i) subject the polyside to start a depolymerization reaction by oxidoreduction, that 3 5 (ü), ei desired, or, we couple the oligoside obtained with a partner of ~ EUILLE.DE REPLACEMENT

W093 / 07178 ~~ j ~~~, ~~ ii; ..

conjugation or a liposome, or, it is incorporated into liposomas, iscoms or cyclo-dextrins and that (iii) the product obtained is mat a suitable pharmaceutical filler.
J Ulla CompO81t1Ot1 preferred pharmaceuticals includes su minus one oligoeide in the form of conjugate.
The pharmaceutical composition according to the invention can be produced from conventional way. In particular we associated an oligoside living room the invention with a thinner or a support acceptable from a point of view pharmaceutical, for example a physiological sauna apyrogetia solution. In outraged, ulie CompU8ltiUn SbIUtl lnVétltlOll may contain ingredients conventional such as a tampon, a preservative or stabilizer, an adjuvant and the case if necessary, a lyophilization excipient. An oligoside salon I'lIIVEIlt1011 can be 1 v CUIl8e1'Vé etl pr esetlCe of a diluent, a support or an ingredient such as previously mentioned. In an alter native way, a thinner, a support or a ingredient can be added just before use.
This is why the invention alternatively proposes a kit.
containing a) a pharmaceutical composition essentially consisting of at least monk an oligoaide according to the invention, with the active agent grade;
b) a composition comprising at least one element selected from a thinner or a support acceptable from a point of view pharmaceutical, a compound having a buffering capacity, an agent preservative or stabilizer and an adjuvant;
c) instructions for concomitant administration eg, in forma mixing the compositions described under a) and b).
A composition according to the invention can be administered by any import which conventional voice, in particular by soue-cutan ~ a, by vola REPLACEMENT SHEET

intramuscular, intravenously, for example under form of suspension for injection or oral.
The administration can take place in single or repeated dose one or more than once after a certain interval. The appropriate dosage varies according to various parameters, by for example, the individual being treated or the mode of administration. We indicates however that good results can be obtained with a unit dose of 1 to 200 ~ g of oligoside in one volume about 0.5 ml.
The invention is presented in more detail in the following examples and with reference to Figures 1 to 4.
Figure 1 shows the unit formulas repetitive of the capsular polysaccharide of S. pneumoniae type 14 (a), type 1 (b), of S. typbi (c), of S. pneumoniae type 6B (d), H. influenzae type B (e), of S. pneumoniae type 19F (f), of N. meningitidis group A (g), of S. pneumoniae type 18C (h), type 23F (i) Klebsiella ozaenae serotype K4 (j), Shigella flexneri serotype lb (k) and Cryptococcus neoformans strain 98 (1). We note that (a) corresponds to a neutral polysaccharide, that (b) and (c) correspond to polysaccharides having acids uronic in the polysaccharide chain, that (d) to (i) correspond to phosphorylated polysaccharides, that (j) and (k) correspond to lipopolysaccharides, respectively anionic and neutral and that (1) corresponds to a polysaccharide anionic fungic.
Figures 2a and 2b show the profiles elution of the non-fragmented polysaccharides of S. typbi (2a) and of S. pneumoniae type 1 (2b) on a 4BCL * Sepharose column.
The chromatography conditions are as follows:
on the Sepharose column * previously balanced with 0.2 M NaCl, 2 ml of a 5 mg / ml polysaccharide solution.
Elution is carried out with 0.2 M NaCl at a rate of 42 ml / h. The elution profile is automatically analyzed in * (trademarks) measuring the optical density at 206 nm. In 2a and 2b, the volume column death is 76.56 ml while the total volume is 201.84 ml.
Figure 3 shows a dextran standard range of 500,000 to 20,000.
Figures 4a and 4b show the profiles of elution of the oligosides obtained by oxidative fragmentation reductive polysaccharides of S. typhi (4a) and S. pneumoniae type 1 (4b) on a Sepharose 4BCL * column. Conditions l0 of chromatography are as previously stated in the description of Figure 2.
Example 1 Fragmentation of the polysaccharide Vi of S. typhi into presence of ascorbic acid.
A dry powder of polysaccharide Vi obtained according to the method of Gotschlich et al, Prog. Immunobiol. Standard. (1972) ~: 485 is taken up in pyrogenic water at a rate of 1 mg / ml.
The average molecular weight (PM) of the polysaccharide corresponds to a KD
20 void on a 4BCL * Sepharose column.
At 500 ml of this preparation preheated to 37 ° C., slowly add, continuously for 2 h, 12.5 ml of a aqueous solution containing 100 mM ascorbic acid, 10 mM
CuS04 and 10 mM FeS04; either a final concentration of acid 0.44 mg / ml ascorbic. The pH of the reaction medium as well obtained is approximately pH 4. The reaction is continued under gentle agitation approximately 3 h (reagent addition time included) at 37 ° C.
The reaction mixture is then filtered through a 30 3K ultrafiltration membrane (cutoff threshold: 3 kg daltons). The retentate is washed a first time with a 0.5 M NaCl solution, then a second time with water. The retentate is taken up in approximately 100 ml of water, then lyophilized for conservation.
* (trademarks) The cleavage rate is of the order of 95 ~ such that calculated by integrating the curves obtained by filtration on Sepharose gel 4BCL * (Figure 4.a). In addition, we estimate the Average PM of the oligoside thus obtained by the Granath method & al (supra). This, expressed as an average elution constant is around 0.6, i.e. a MW of 60,000 EqD (taking for dextran reference).
Analysis of the oligoside by NMR spectrometry (nuclear magnetic resonance) clearly indicates that the l0 structure of the repeating unit characteristic of polysaccharide a been kept after fragmentation. In particular, there is no loss of branched functional groups or sugars of the polysaccharide skeleton.
The characteristic chemical functions of the unit of the Vi polysaccharide are analyzed by assay color. The branched 0-acetyl group is assayed by the method of Hestrin, Biol. Chem. (1949) 1_88:
249 while the polyacid of the linear structure is dosed by the method of Stone et al, Clin. J. Microbiol. (1988) ~:
719. The assays are carried out in parallel on polysaccharide Vi not fragmented and on the oligoside formed.
The [O-acetyl] / (polyacid] ratios measured for the polysaccharide and oligoside are identical. This shows that the polysaccharide fragmentation method made it possible to conserve all of the labile O-acetyl groups.
Example 2: Fragmentation of the polysaccharide Vi of S. typbi in the presence of hydrogen peroxide (H202).
A dry powder of the polysaccharide Vi obtained according to the method of Gotschlich et al, (supra) is repeated in buffer phosphate 0.2 M pH 7.5 at 0.4 mg / ml. PM of polysaccharide corresponds to a zero KD on a Sepharose column 4BCL *.
* (trademark) At 100 ml of this preparation preheated to 37 ° C., slowly add, with gentle stirring, 11 ml of a solution aqueous containing 0.3 mg / ml of H2O2 and 1.1 ml of a solution of CuS04 at 2.6 mg / ml. The reaction is continued with stirring gentle about 1 hour at 37 ° C.
The experience is repeated under the same conditions with the exception of the reaction time which is 2 hours instead a.
In both cases, each oligoside thus obtained is then collected and purified as described in Example 1.
Likewise, the characteristics of oligosides as well obtained are highlighted according to the methods cited in Example 1. The results are as follows:
- In both cases, the rate of cleavage of the polysaccharide Vi is of the order of 100ô.
- The average PM of the oligosides formed after one and two hours corresponds respectively to a KD of in the range of 0.7 and 0.8; i.e. 30,000 and 10,000 EqD.
- In both cases, there is no evidence of modification in the structure of the unit repetitive.
xempe 3: Fragmentation of the polysaccharide of N. meningi-tidis group A
A dry powder of N. meningitidis polysaccharide group A as obtained by the method of Gotschlich et al (supra) is taken up in 0.2 M phosphate buffer pH 7.5, i.e.
1 mg / ml, i.e. 5 mg / ml. The average PM of polysaccharide corresponds to a KD of 0.15 as measured on a Sepharose column 4BCL *.
The preparations at 1 and 5 mg / ml are preheated to 37 ° C.
3a) To 100 ml of the preparation at 1 mg / ml, the following is added 0.75 ml of a reaction solution containing * (trademark) 16a 100 mM ascorbic acid, 10 mM CuS04 and 10 mM of FeS04. The reaction is continued with stirring for 1 h 30 to 37 ° C. Then again 0.75 ml of the reaction solution; we thus obtain a final concentration of ascorbic acid of 0.26 mg / ml. The reaction is continued again for 1 h 30 under the same conditions.
3b) To 100 ml of the preparation at 5 mg / ml, the following is added 1.5 ml of a reaction solution containing _ _. . ___L_ ____ '~ ~ s.' s ,; ~ e,.

mM da CuSO, and 10 mM dc FeSO,; either a concentration final ascorbic acid of 0.26 mg / ml. The reaction is continued with stirring 1 h 30 at 37'C.
5 The oligosidea obtained in 3a) and 3b) are collected and purified as described in Example 1. Likewise, the analyzes are implemented such that mentioned in Example 1. In both cases, we observe a cleavage rate 100%, cane deterioration of the structure of the repetitive unit as NMR analyst. The average PM of the oligoside obtained in 3a) is therefore expected to 10 Ka 0.7 (i.e. 30,000 EqD) while the average PM of the oligoside obtained in 3b) corresponds to a Kn of the order of 0.4 (or 110,000 EqD).
All of these results indicate that by varying the conditions experentalental (concentration of polysaccharide and oxidative reagent-r eductor as well as the mode of addition of the reactive agent and the reaction time) can obtain substantially different mean PM oligosides while retaining a 100% cleavage rate.
Finally this example as well as Example 2, show that it is fragmentation by oxidoreduction and not by acid hydrolysis or basic insofar as this fragmentation can be implemented at a pH close to neutral.
EXAMPLE 4 Fragmentation of the Polysaccharide of S. Pneumonia of typas 1 and 19F and H influenzas type B, in the middle buffered.
Example 3 (3a and 3b) is repeated using the polyoaidea of S.
pmeumanisa types 1 and 19F as obtained according to the method described in the French patent application FR 2 495 939 published on June 18, 1982 as well as the polyacid of H infuanzaa type B obtained according to the method of Gotachlich et al (supra);
REPLACEMENT FEiJILI., E

WO 93/07178 PCT / FR92 / 0095 ~
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<. . ; .ï.t ~

The results are as follows Protocol a) Protocol b) i ~ x lOJCO
of s ~~ e pole ex .o ~ n ae r ~ px .onA ~ r ~ ~
ae ie ~ ri ~ l ~ d ~ rte Pdi ~ adde elive ~ e in e ~) S.pneu ~ oci ~ O, C ~ 0.8 ~ 100 0.3 ~ 100 S.paeusoaiae0,0! 0.80 100 0.3 100 H, intluenraeO, i O, TS 100 11D liD
B

ND: not determined Analysis of the oligoside by NMR spectrometry (magnetic resonance nuclear) clearly indicates that the structure of repeating units polyoeid characteristics was retained after fragmentation.
Example 5: Fragmentation of polysaccharides N. menin ~ itidis group A and S. praaumonise types 1, 14, IBC, 19F and 23F eaa 2 5 non-buffered medium.
The polyoeid preparations as well as the reactions are set to aa ~
work as described in Examples 3 and 4 with exceptions following: (i) the polysaccharide preparations are prepared at 5 mg / ml in water nonpyrogenic, (ü) the reaction solution is added a single pea to a few final concentration of 2.5 mg / ml and (iii) the reaction is continued only for 1 hour.
The oligosides obtained are collected and purified as described in Example 1. Their characteristics are assessed SUBSTITUTE SHEET

t ~ l ~ tk ~ '~ 5 ~ ~'; ,.
.L

as mentioned in Example 1. The cleavage rate of each of the polysaccharides is 100% without there being deterioration of the structure of repeating units. This checkerboard point was also m18 in evidence by colorimetric assay: The hexosea are assayed by the method of Scott et al, Anal. Chem. (1953) 25: 1650, hexosaminea by the method of Gatt et al, Anal. Biochem. (1965) 15: 167, rhamnose by the method of Dische et al, J.
Biol. Chem. (1948) 175: 595 and phosphate by the method of Chen et al, Anal. Chem. (1956) 28; 1? 56. Regarding the polysaccharide of S. pneumonia type 14 and the oligoside derived from it by fragmentation, reports [hexose] / (hexosamine] are substantially identical. The same is true for ratios (rhamnose] / [hexoae] and (phosphate] / (hexose] respectively considered in the case of S. pmeumvuiae types 18C and 23F.
NMR spectrometry analysis of oligosidea does not highlight of signals indicating a heterogeneity o ~ enéité dakl8 the repetitive r units and goes up e as well as oligoaidea consist of an intact repeating unit.
Finally, with regard to the average PM of the oligosides, the results are as follows Medium PM strain Average PM
of polyoeide oligoside (expressed in expressed in KB) Ku) N.mer ~ in ~ itidis 0.15 0.66 AT

Pneumvnise 0.04 0.72 S.pnaumvnise 0.01 0.72 S.pmeumoi ~ isa0.03 0.75 S.pmaumomisa 0.04 0.67 S.pnaumonisa 0.07 0.57 REIV SHEET ~ PLACEMENT

.: ~~, ". ~~ .1 ~~ ..

Example 6 Preparation of an Oligoside Vi Conjugate of S. typbi /
cholera toxin B subunit.
Ga) Introduction of an N & group on oligoside V ;.
A lyophilieat of the oligo-aid Vi as obtained in Example 1 is r taken in a phosphate buffer pH 8 to r Bison of 10 mg / ml. At this 10 prparation, diaminohexane and NaCNBIi ~ are added in solutions;
each one has a final concentration of 12.5 mg / ml. The reaction is continued during G
days at ambient temperature. The preparation is then dialyzed against water and lyophilized.
15 By assay -selozi the method of Shnyder et al, Anal. Biochem. (1975) G4 284, it is said that groups N & have indeed been introduced. Likewise, by assay according to Heatrin, it is shown that the 0-acetyl groups are always retained.
20 6b) Introduction of a reactive function.
The lyophilisate obtained in 6a) is taken up in solution at the rate of 40 mg / ml. To 20 ml of this preparation, 80 ml of a 40 mg / solutiota are added.
ml of diauccinimidyl suberate (DSS) to dimethyl sulfoxide (DMSO). The reaction is continued for one hour at room temperature. The preparation is then precipitated in a mixture of DMSO / dioxane.
6c) Canjugaison.
The precipitate obtained in 6b) is taken up in a 0.5 M NaCl solution.
to r Bison of 40 m ~ / ml. In by allele, we prepare a solution of soue-unit B
cholera toxin - (Tayot et al, Eur. J. Biochem. (1981) 113: 249) in 0.2 M phosphate buffer pH G, 5 at 10 mg / ml.
3 5 The two solutions thus prepared are mixed REPLACEMENT FEUtLLE_DE

R t ~~. ~. ..x.
~ '.r. ~: .5 1. .

ellsemble. The weighted ratio of oli ~ oside / protein is approximately 1. The r reaction Eat at room temperature for 15 hours.
6d) PurifiCatioll.
The mélsn ~ e is then filtered through a ultrafiltration kit (Millipore) equipped with a saw frame with a separation threshold of 5.10 daltone, in order to eliminate prUtélYl and U11 ~ U61dé 11U11-Couplé. The retentate East washed in NaCI 0.3 M pul8 CoYlserVé at ral8oti of 200 u ~ / ml in NsCl 8 / 1.000, mcrtlüolaté 1 / 10,000, at 4'C, after sterile filtration.
Example 7: Preparation of oli ~ oside / atiatoxin conjugates toxoid.
Oti eonju ~ ue oli ~ osides as obtained from polyacids S. pnaumvniaa types 14 and 23F in 'Example 5, as well as the oli ~ oside obtained at starting from the polyoeide Vi of S.typl: i in Example 1, operating in a manner analogous to the rest otbcole pr in Example 6 ~ and by replacing the sub-unit B of cholera toxin by tetanus toxoid prepared according to the method de Mueller and Milles, J. Bact. (1954) 67: 67I.
The conjugates obtained from oliosides of S. p17eu711JlIi8a are canned at 250 u ~ / ml.
EXAMPLE 8 Demonstration of the Immunogenic Power of Canju ~ SIU.
OF1 mice (Iffa Credo) are distributed in lots of 8. Each mouse of a batch receives 0.5 ml of one of the solutions prepared in Examples 6 and? or in parallel, 0.5m1 of the corresponding native polysaccharides. Injections take place subcutaneously. These injections are repeated 14 days apr2 ~ s. In not light, a control batch receives only water Physiology ~ iqud.
14 to 28 days after the pl'eml ~ re 111jeCti011, we take a sample 3 5 blood and titi e las anticos ps I ~ G anti-polyoeide by Elisa assay SHEET ~ E REisAPLAGE (~ 61ENT

WO 93/07178 PCf / FR92 / 00955, t: Zf ~ '~, ~ ~ 1 ~.

(the titrâ ~ e plates are covered with non-fragmented polyoaide). The rate antibody at 14 and 23 days shows that the conjugates are immuno ~ èiies. In in each case, the rate of anticor ps is higher than that induced by the native polysaccharide spying horn. In addition there is a rebound effect characteristic of a T-dependent anti ~ acne.
Example 9: Vaccine pharmaceutical composition comprising conjugated as active teens.
1Q Le6 CUIljtt ~ ué8 oll ~ oBldeB Vi of S. typhi / tetanus toxoid obtained eelokl Example 7 BUIlt fUI'mu168 in a vaccinated dose of 0.5 ml intended at be injected into humans subcutaneously. Composition per dose being - Oli ~ osides Vi of S. typhi conjugates 10 ~ a ~
- Phosphate buffer at pH 7,475 u ~ (eri ions POv) - NaCl 4250 ~ g - yesterday thiolate 0.05 ~ r ~
- Water ppi qs 0.5 ml REPLACEMENT SHEET

Claims (54)

1. Process for the preparation of an oligoside by depolymerization of an antigenic polysaccharide derived from an agent pathogen, the repeating units of which contain at least a labile chemical function, retaining the structure repetitive units with their labile functions, said labile chemical functions being defined as functions which, when the polysaccharide is subjected to a process for depolymerizing polysaccharides by fragmentation ultrasound or by acid or base hydrolysis, undergo a modification altering antigenicity, wherein:

(i) subjecting said polysaccharide to a reaction oxidation-reduction depolymerization, and (ii) the oligoside thus obtained is collected. 2. Method according to claim 1, charac-terized in that the reaction of redox depolymerization in the presence of an agent redox which is ascorbic acid or peroxide of hydrogen. 3. Method according to claim 2, character-ized in that the reaction of redox depolymerization in the presence of a salt iron or copper. 4 . Process according to any one of the claims cations 1 to 3, characterized in that one implements the oxidation-reduction depolymerization reaction on a aqueous solution containing from 0.5 to 50 mg/mL of said polysaccharide. 5. Process according to claim 4, in which said solution contains: from 0.5 to 10 mg/mL of said polysaccharide. 6. Method according to claim 4 or 5, characterized in that said solution has a pH of 3 to 8.5. 7. Process according to claim 6, in which said solution has a pH of 3 to 6.5. 8. Process according to any one of the claims cations 1 to 7, characterized in that the oxidation-reduction depolymerization reaction at a temperature from 4 to 60°C. 9. A method according to claim 8, wherein which said temperature is in the range of 1.8 to 40°C. 10. A method according to any one of claims cations 1 to 9, characterized in that the oxidation-reduction depolymerization reaction in presence of an oxido-reducing agent which is the acid ascorbic. 11. Process according to claim 10, characterized ized in that ascorbic acid is used at a concentration of 0.01 to 25 mg/mL. 12. Process according to claim 11, in which ascorbic acid is used at a concentration from 0.1 to 10 mg/mL. 13. Method according to any one of the claims cations 10 to 12, characterized in that said reaction in the presence of metallic iron salts. 14. Process according to any one of the claims cations 10 to 12, characterized in that said reaction in the presence of copper metal salts. 15. A method according to any one of claims cations 10 to 12, characterized in that said reaction in the presence of metal salts of iron and copper. 16. A method according to any one of claims cations 13 to 15, characterized in that the said salts metals are present at a concentration of 1 mM at 100nM
. 17. Process according to claim 16, in which said metal salts are present at a concentration of 1 to 10 mM. 18. Process according to any one of the claims cations 1 to 9, characterized in that the oxidation-reduction depolymerization reaction in presence of an oxido-reducing agent which is peroxide of hydrogen. 19. Process according to claim 18, characterized ized in that hydrogen peroxide is used to concentration from 0.1 to 100 mM. 20. A method according to claim 19, in which hydrogen peroxide is used at a concentration of 0.5 to 10 mM. 21. A method according to any one of claims cations 18 to 20, characterized in that said reaction in the presence of metallic iron salts. 22. Process according to any one of the claims cations 18 to 20, characterized in that said reaction in the presence of copper metal salts. 23. Process according to any one of the claims cations 18 to 20, characterized in that said reaction in the presence of metal salts of iron and copper. 24 . Process according to any one of the claims cations 1 to 23, characterized in that said function labile chemical is chosen from the groups phosphate, acetyl and pyruvate. 25. Process according to claim 24, characterized ized in that said labile chemical function is a phosphate group. 26. Process according to claim 24, characterized ized in that said labile chemical function is a acetyl group. 27. Process according to claim 24, characterized ized in that said labile chemical function is a pyruvate group. 28. A method according to any one of claims cations 1 to 27, characterized in that said polysaccharide antigenic is a capsular polysaccharide derived from a bacterium pathogenic. 29. Process according to claim 28, characterized ized in that said pathogenic bacterium is chosen from bacteria belonging to the genera Staphylococcus, Streptococcus, Kiebsiella, Salmonella, Escherichia, Neisseria and Haemophilus. 30. Process according to claim 29, characterized ized in that said pathogenic bacterium belongs to the genus Staphylococcus. 31. Process according to claim 29, characterized ized in that said pathogenic bacterium belongs to the genus Streptococcus. 32. Process according to claim 29, characterized ized in that said pathogenic bacterium belongs to the genus Klebsiella. 33. Process according to claim 29, characterized ized in that said pathogenic bacterium belongs to the genus Salmonella. 34. Process according to claim 29, characterized ized in that said pathogenic bacterium belongs to the genus Escherichia. 35. Process according to claim 29, characterized ized in that said pathogenic bacterium belongs to the genus Neisseria. 36. A method according to claim 29, characterized in that said pathogenic bacterium belongs to the genus Haemophilus. 37. Process according to claim 29, characterized ized in that said pathogenic bacterium is Salmonella typhi. 38. A method according to claim 29, wherein which said pathogenic bacterium is Streptococcus pneumonia. 39. A method according to claim 29, wherein which said pathogenic bacterium is Neisseria meningitis. 40. A method according to claim 29, wherein which said pathogenic bacterium is Haemophilus influenzae. 41. Method according to one of claims 1 to 40, characterized in that it further comprises the step consisting (iii) either in coupling the oligoside obtained in (ii) to a conjugation partner or carrier to obtain said oligoside in the form of a conjugate or bond of covalently to a carrier, or to incorporate the oligoside obtained in (ii) in a vector. 42. Process according to claim 41, characterized ized in that said step (iii) consists in coupling the oligoside obtained in (ii) to a conjugation partner. 43. Process according to claim 42, characterized ized in that said conjugation partner is a peptide, protein or organic polymer. 44. Process according to claim 43, characterized ized in that said protein is a protein bacterial. 45. Process according to claim 44, characterized ized in that the bacterial protein is a toxin, a toxoid, a subunit of a multimeric toxin, a membrane protein or a subunit of a membrane protein multimeric membrane. 46. Process according to claim 41, characterized ized in that said step (iii) consists in coupling the oligoside obtained in (ii) to a carrier. 47. Process according to claim 46, characterized ized in that the carrier is a liposome. 48. Process according to claim 41, characterized ized in that said step (iii) consists of incorporating the oligoside obtained in (ii) in a vector. 49. Process according to claim 48, characterized ized in that the vector is chosen from liposomes and the ISCOMS. 50. Oligoside obtained by the process of one any of claims 1 to 49. 51. Pharmaceutical composition comprising at title of active agent at least one oligoside obtained by the method according to any one of claims 1 to 49 and a pharmaceutically acceptable excipient. 52. Composition according to claim 51, characterized in that it constitutes a composition vaccine. 53. Use of an oligoside obtained by the method according to any one of claims 1 to 49, as an active ingredient of a pharmaceutical composition intended to strengthen the immune defenses of a individual. 54. Use according to claim 53, characterized in that the composition is intended to perform a vaccination.